This invention relates to compositions for inactivating viruses that can be used as diluents for preparing immunoassay specimens. These inactivating compositions have the additional advantageous characteristics of improving both the selectivity and sensitivity of tests based on the antibody-antigen reactivity of the viruses.
Enzyme-linked immunosorbent assays (ELISA) are well-known immunoassays that provide a routine means for detecting antibodies to specific antigenic determinants, including those found on viruses, which are particularly useful in screening large numbers of samples. In these tests an antigen is immobilized on a solid support, and specimens that may contain the respective antibody are placed in contact with the immobilized antigens. Any unobstructed antibodies that are present in the specimens become bound to the fixed antigens. As the bound antibodies contain immunoglobulin characteristic of their source, e.g., human serum antibodies contain human immunoglobulin, the bound antibody is detected by contacting the solid phase medium with "anti-source" immunoglobulins, e.g., anti-human immunoglobulins, that are linked to a detectable tag, such as an isotope or an enzyme. After contacting the solid phase with a specimen and washing the solid phase, the detection of the tag on any solid phase sample indicates the presence of the antibody. The same principles apply in immunoassays that immobilize antibodies in the solid phase and detect antigens present in test specimens.
Immunoassay tests, being highly sensitive, are conducted using diluted specimens. Dilution aids in eliminating or reducing interference that may be caused by other components of the specimen. Commonly used diluents comprise water, serum, buffered saline and preservatives and do not inactivate pathogenic organisms or viruses. As a result, substantial quantities of diluted specimens, wash solutions and solid substrates are produced during the course of conducting ELISA and other immunoassay tests, the handling and disposal of which becomes a significant problem as large quantities of waste liquids are produced that could contain infectious agents. For example, in large-scale assays of blood and blood products for viruses such as Human T-Lymphotropic Virus Type III (HTLV-III) and Hepatitis virus, the tested specimens, the waste solutions and the used solid media could be sources of infection for laboratory workers and others. Accordingly, a means for inactivating any pathogenic virus that may be in the specimens tested is of great importance for eliminating a significant health hazard.
The inactivation of HTLV-III with reagents and methods commonly used in virology laboratories have been studied by several investigators. Spire et al reported the inactivation of HTLV-III with sodium hypochlorite, .beta.-propionolactone, formalin, glutaraldehyde, formalin and ethanol by incubating the virus with various concentrations of each disinfectant for one hour and assessing inactivation by analyzing for reverse transcriptase activity (The Lancet, Oct. 20th, 1984). Spire et al, in a different article, reported the results of inactivation tests using heat, gamma radiation and ultraviolet radiation (The Lancet, Jan. 26th, 1985), in which inactivation was only achieved at relatively high temperatures and radiation levels. Martin et al also reported inactivation using conventional laboratory disinfectants and detergents (The Journal of Infectious Diseases, Volume 152, No. 2, August 1985). All of the chemical agents tested by these investigators caused inactivation at certain concentrations, except for Tween-20.TM. detergent. With the exception of the Tween-20.TM. and Nonidet P-40 detergents, all of the chemical agents reviewed by these investigators were known to be strong disinfectants. It was reported that Nonidet P-40 (NP-40) provided adequate inactivation at a concentration of 1.0 weight percent. Tween-20.TM., however, did not prove to be an effective inactivating agent, even at concentrations as high as 2.5%.
In virology laboratories it is common to use high concentrations of detergents and salt together for inactivating viruses, as detergents are known to remove the protein envelope and the salt causes the nucleotide-protein core to dissociate. Such media have not been used as diluents for preparing specimens of, for example, serum, blood, plasma or urine, however, because concentrations of detergents and salt sufficient to render a virus inactive would have been expected to interfere with the analytical results. For example, NaCl is known to precipitate ribonucleic acids. Moreover, antigen-antibody reactions are ionic and high concentrations of salt would theoretically affect the surface charges on the antigen and antibody molecules, thus interfering with the formation and stability of the ionic bonds. On the other hand, diluent compositions containing a large proportion of normal serum and relatively low concentrations of salt and detergent would be expected to be satisfactory diluents but not effective virus inactivators.
It was an object of the present invention to provide diluent solutions that could be used in preparing specimens for immunoassay analysis that would act effectively to inactivate any virus present while not reducing the specificity and sensitivity of the tests.